The goal of this proposal is to develop cell-based therapies that lead to the better healing of traumatic head injuries. Our first strategy will be to use genetics and embryology in zebrafish to identify factors that can convert human embryonic stem cells into replacement skeleton for the head and face. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. As zebrafish develop rapidly and can be grown in large numbers, a growing number of researchers are using zebrafish to study how and when cells decide to make a specific type of tissue – such as muscle, neurons, and skeleton - in the vertebrate embryo. Recently, we have isolated two new zebrafish mutants that completely lack the head skeleton. By studying these mutants, we hope to identify the cellular origins and genes that make head skeletal precursors in the embryo. These genes will then be tested for their ability to drive human embryonic stem cells along a head skeletal lineage. Our second strategy will be to test whether a population of cells, similar to the one that makes the head skeleton in the embryo, exists in the adult face. We have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation. In this proposal, we use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can give rise to new head skeleton in response to injury. Traumatic injuries to the face are common, and treatment typically involves grafting skeleton from other parts of the patient to the injury site. Unfortunately, the amount of skeleton available for grafts is in short supply, and surgeries often result in facial disfigurement that causes psychological suffering for the patient for years to come. Here we propose two better treatments that would lead to more efficient healing and less scarring. The first treatment would be to differentiate human embryonic stem cells, a potentially limitless resource, into skeletal precursors that can be grafted into the head injury site. By understanding the common pathways by which head skeletal cells are specified in the zebrafish embryo and human embryonic stem cells, we will be able to generate skeletal replacement cells in large quantities in cell culture. The second treatment would be to stimulate adult stem cells already in the face to regenerate the injured head skeleton. If successful, our experiments on zebrafish jaw regeneration will allow us to devise strategies to augment the natural skeletal repair mechanisms of humans.

Statement of Benefit to California:

Traumatic injuries to the head, such as those caused by car accidents and gunshot wounds, are commonly seen in emergency rooms throughout California. Current treatments for severe injuries of the head skeleton involve either grafting skeleton from another part of the body to the injury site or, in cases where there is not sufficient skeleton available for grafts, implanting metal plates. Although these operations save lives, they often result in facial disfigurement that causes psychological suffering for the patient for years to come. For this reason, there is enormous interest in cell-based skeletal replacement therapies that will heal the face without leaving disfiguring scars. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. Thus, we are using the zebrafish embryo to rapidly identify factors that can make head skeletal precursors, and then asking if these same factors can instruct human embryonic stem cells to form skeletal replacement cells. In addition, we have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation, and we will use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can regenerate the face. The successful completion of these experiments would allow us to both generate unlimited amounts of head skeletal precursors for facial repair and stimulate latent skeletal repair mechanisms. The combination of these approaches will lead to therapies that promote a more natural healing of the face, thus allowing Californians to eventually resume normal lives after catastrophic accidents.

Progress Report:

A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head. An understanding of how these skeletal cells form in the embryo will aid in our long-term goal of producing skeletal replacement cells in culture and stimulating new skeleton to form after traumatic head injuries.

In the first year of this award, we have made a landmark discovery concerning how the skeletal-forming cells arise in the head. The head skeleton arises from an unusual cell population called the neural crest, which is characterized by its ability to form a very wide diversity of cell types in the embryo. We had previously described the isolation of two mutant lines of zebrafish that completely and specifically lack the head skeleton. We have now identified a mutation in a variant histone protein as the genetic basis for the lack of head skeleton in one of these lines. Histone proteins play a central role in not only wrapping our DNA but also controlling which genes are active in different cell types. Despite the fact that histones are expressed in every cell in the body, we have found that variant histones are uniquely required for neural crest cells to acquire skeleton-forming potential. In particular, our findings indicate that the head skeleton forms by a process of developmental reprogramming, in which precursor cells with a limited potential undergo large-scale changes in their DNA packaging such that they are able to form a much wider array of cell types.

Controlled cell reprogramming is becoming one of the most promising directions of regenerative medicine. For example, there is enormous therapeutic potential in being able to take cells from a patient, reprogram these cells back to a naïve state with defined factors, and then induce these cells to form replacement cells of any type that can be introduced back into the patient. Our discovery that the vertebrate embryo uses a similar process of reprogramming to generate head skeletal cells during development provides us an opportunity to better understand how reprogramming works at a molecular level. In addition, our finding that head skeletal cells form by developmental reprogramming suggests that adult patient-specific cells can be directly reprogrammed to form head skeletal precursors. Moreover, as the variant histone we have identified is identical at the protein level between zebrafish and humans, it is likely that the reprogramming process we have discovered operates in humans as well. Thus, in the coming years of this grant we are excited to develop methods of reprogramming adult patient-specific cells to a head skeletal fate, such that we can generate large quantities of replacement cells to repair the face and skull.

A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head. An understanding of how these skeletal cells form in the embryo will aid in our long-term goal of producing skeletal replacement cells in culture and stimulating new skeleton to form after traumatic head injuries.

In the second year of this award, we have extended our initial findings that variant histones play an essential role in the generation of head skeletal cells. Skeletal cells usually arise from a cell population called the mesoderm. However, in the head a unique population of ectoderm cells, which normally forms derivatives such as neurons and skin, has an added ability to form skeletal cells. How these cranial neural crest cells acquire this extra potential remains unclear. Here we show that variant histones function within the ectoderm to give cranial neural crest cells skeletal-forming ability. In addition, we have used biochemical analysis in a human cell line to demonstrate how mutations in a particular H3.3 type of variant histone disrupt the association of histones with DNA. Furthermore, we have developed tools which allow us to analyze how variant histone changes throughout the genome endow neural crest ectoderm cells with mesoderm-like skeletal-forming potential.

Variant histones have been implicated in cell reprogramming, whereby a mature cell regains the ability to form many more cells that can repair a damaged tissue. As we find that variant histones are required for reprogramming of the ectoderm to form neural crest during early development, we believe that we can use this pathway to convert patient-specific cells to neural crest and skeletal cells that can be used for facial repair. To test this, we have developed a mouse model in which we can detect the ability of neural crest genes to convert mature cells to neural crest and skeletal fates. In parallel, we have developed a model of jaw regeneration in zebrafish that will allow us to test whether adult cells within the animal can be reprogrammed to repair the skeleton in response to injury. During this period, the generation of transgenic tools has allowed us to begin to address which cell types can give rise to new skeleton in response to injury. In the coming years, we hope that our work in model systems will lead to therapies for head skeletal injuries on two fronts: the generation of large amount of head skeletal precursors from patient-specific cells and the induction of increased regenerative ability of cells within the patient.

A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. We have previously described that zebrafish with a mutant form of a variant histone H3.3 protein have very specific defects in the ability of neural crest cells to form skeleton. As histone H3.3 is a core component of the chromatin around which DNA is wrapped, our findings suggest a novel mechanism by which changes in chromatin structure endow the neural crest with the ability to form a wide array of derivatives. In the third year of this award, we have expanded our analysis to examine how the incorporation of histone H3.3 is regulated specifically in the neural crest population. Our data suggest that such H3.3 incorporation may depend on a novel chaperone protein as reducing the function of several known H3.3 chaperone proteins does not lead to specific neural crest or head skeletal defects. A clue to what regulates H3.3 activity comes from a second zebrafish mutant – called myx - that we are studying, which has head skeletal defects similar to what we see in our H3.3 mutant. We have mapped the myx mutant interval to a very small region that contains a putative H3.3 chaperone protein. We are currently establishing whether loss-of-function of this chaperone accounts for myx defects. Together, our studies of H3.3 and myx mutants will shed light on how to generate cells with the ability to form replacement head skeleton in patients. To this aim, we have also begun experiments to use our findings in zebrafish to directly convert mammalian cells (initially mouse but then in humans) to a neural crest and skeletal fate.

A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. We have a limited ability to repair defects in our skeleton, for example after bone fracture. However, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jaw following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that the zebrafish lower jaw bone regenerates through a cartilage intermediate, in contrast to the direct differentiation to bone during development. Hence, our findings indicate that bone regeneration in zebrafish is a cellularly distinct mechanism than bone development. Furthermore, we have found that the FGF signaling pathway is greatly upregulated during early jaw regeneration. FGF signaling also mediates the regeneration of the heart and other organs in zebrafish, and thus jaw regeneration may rely on a common regenerative program throughout the zebrafish. In the coming period, we plan to test the functional requirements of FGF signaling in mediating jaw regeneration, as well as identifying the stem cell populations that are the FGF-dependent source of new bone.

A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. In the previous grant cycle, we published a manuscript in PLoS Genetics describing the role of a variant histone H3.3 protein in controlling the ability of neural crest cells to form the head skeleton of zebrafish. As histone H3.3 is a core component of the chromatin around which DNA is wrapped, our findings suggest a novel mechanism by which changes in chromatin structure endow the neural crest with the ability to form a wide array of derivatives. In addition, we published a separate study in PLoS Genetics that showed a critical role of Twist1 in guiding these neural crest cells to make head skeleton at the expense of other cell types such as neurons. Together, our studies of H3.3 and Twist1 in zebrafish will shed light on how to generate cells with the ability to form replacement head skeleton in patients. In ongoing experiments, we are using principles from our zebrafish system to directly convert mammalian cells (initially in mouse but then in humans) to a neural crest and skeletal fate.

A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. We have a limited ability to repair defects in our skeleton, for example after bone fracture. However, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jaw following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that during zebrafish lower jawbone regeneration, an unusual cartilage intermediate is able to directly make replacement bone, which is in marked contrast to the way bone is made during development. Furthermore, we have found a potentially critical role of the Ihh signaling pathway in allowing regenerating cartilage cells to directly make replacement bone. In the coming period, we plan to test the functional requirements of Ihh signaling in mediating jaw regeneration, as well as identifying the cellular source of bone-producing cartilage cells during jaw regeneration. As similar bone-producing cartilage cells may also be present in human fractures, lessons learned from zebrafish may allow us to stimulate these cells and hence augment bone repair in patients.

A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. We had previously identified a unique role of histone replacement within the neural plate precursor cells that allows neural crest to make skeletal derivatives. In the current grant cycle, we now find that misexpression of groups of neural plate transcription factors is able to convert cells to a neural crest fate, and we are currently exploring whether this occurs by stimulating the histone replacement program we found to be so important for neural crest development. We are also testing whether similar neural plate transcription factors can convert mammalian cells to a neural crest fate, with the eventual goal to use this technique to generate an unlimited supply of patient-specific bone and cartilage replacement cells for skeletal repair.

A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. While we have a limited ability to repair defects in our skeleton, for example after bone fracture, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jawbone following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that during zebrafish lower jawbone regeneration, cartilage cells are able to change their fate to directly make replacement bone, which is in marked contrast to the way bone is made during development. We have also identified a critical role of the Ihh signaling pathway in bone regeneration and in particular the generation of the critical cartilage intermediate. In adults lacking the Ihha protein, no cartilage forms after jawbone amputation and the jawbone fails to heal properly. Moreover, in collaborative work we find that such bone-producing cartilage cells may also be present in a mammalian model of bone healing. We are therefore excited by the prospects of using similar bone-producing cartilage cells to repair large skeletal wounds in patients.